Feline immunodeficiency virus is a lentivirus of domestic cats that causes significant lifelong infection. Infection with this or similar lentiviruses has been detected in several nondomestic feline species, including African lions (Panthera leo). Although lion lentivirus (FIVple) infection is endemic in certain lion populations in eastern and southern Africa, little is known about its pathogenic effects or its epidemiological impact in free-ranging lions. This report describes the epidemiological investigation of lentivirus positivity of free-ranging lions in the Kruger National Park, South Africa. A nested polymerase chain reaction assay for virus detection was performed on all whole blood samples collected. In addition, serum samples were tested for cross-reactive antibodies to domestic feline lentivirus antigens and to puma lentivirus synthetic envelope peptide antigen. The results were analysed in conjunction with epidemiological data to provide a descriptive epidemiological study on lion lentivirus infection in a free-ranging population of lions. The overall prevalence of lentivirus infection was 69%, with a prevalence of 41% in the north of the park, and 80% in the south. Adult males had the highest prevalence when combining the factors of sex and age: 94%. The lowest prevalences were found among juveniles, with male juveniles at 29%. Adults were 5.58 times more likely to test positive for FIVple than juveniles, with adult males being 35 times more likely to be test positive for FIVple compared with juvenile males. This research represents the 1st epidemiological study of the lion lentivirus among free-ranging lions in the Kruger National Park.